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It is important to use good, adequate, & industrially usable medium. Enhances harness of the organism’s full industrial potentials. If media was not suitable, the production of the desired product will be reduced & toxic materials may be produced. Liquid media are generally employed in industry because they require less space. LM are more amenable to engineering processes, and eliminate the cost of providing agar and other solid agents.

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THE BASIC NUTRIENT REQUIREMENTS OF INDUSTRIAL MEDIA For industrial or for laboratory purposes, media must satisfy the needs of C, N, minerals, growth factors, and water. No inhibitory materials. Complete analysis of the organism’s nutrients needs should be performed. C or energy requirements are usually met from carbohydrates (glucose, starch or cellulose & …., etc). Energy sources may include hydrocarbons, alcohols, or even organic acids.

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In formulation industrial medium, the carbon content must be adequate for the production of cells. For most organisms the weight of organism produced from a given weight of carbohydrates (known as the yield constant) under aerobic conditions is about 0.5 gm of dry cells per gram of glucose. Carbohydrates are at least twice the expected weight of the cells and must be put as glucose or its equivalent compound.

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Nitrogen is a key element in the cell. Most cells would use ammonia or other nitrogen salts. For bacteria the average N content is 12.5%. To produce 5 gm of bacterial cells per liter would require about 625 mg N (Table 4.1). Any nitrogen compound which the organism cannot synthesize must be added. Minerals form component portions of some enzymes. The major minerals needed include P, S, Mg and Fe. Trace elements: manganese, boron, zinc, copper and molybdenum. Growth factors include vitamins, amino acids and nucleotides and must be added to the medium if the organism cannot manufacture them.

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CRITERIA FOR THE CHOICE OF RAW MATERIALS USED IN INDUSTRIAL MEDIA Cost of the material Ready availability of the raw material Transportation costs Ease of disposal of wastes resulting from the raw materials Uniformity in the quality of the raw material and ease of standardization Adequate chemical composition of medium Presence of relevant precursors Satisfaction of growth and production requirements of the microorganisms

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Cost of the material The cheaper the raw materials the more competitive the selling price of the final product. Lactose is more suitable than glucose in some processes (e.g. penicillin production) because of the slow rate of its utilization, it is usually replaced by the cheaper glucose. The raw materials used in many industrial media are usually waste products from other processes. E.g., Corn steep liquor and molasses.

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Ready availability of the raw material If it is seasonal or imported, then it must be possible to store it for a reasonable period. The material must be capable of long-term storage without change in quality. Transportation costs The closer the source of the raw material to the point of use the more suitable it is for use, if all other conditions are satisfactory. Ease of disposal of wastes resulting from the raw materials The disposal of industrial waste is rigidly controlled in many countries.

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Waste materials often find use as raw materials for other industries. Thus, spent grains from breweries can be used as animal feed. But in some cases no further use may be found for the waste from an industry. Its disposal could be expensive. When choosing a raw material therefore the cost, if any, of treating its waste must be considered.

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Uniformity in the quality of the raw material and ease of standardization Composition must be reasonably constant in order to ensure uniformity of quality in the final product and the satisfaction of the customer. E.g., molasses as waste product of sugar industry. Each batch of molasses must be chemically analyzed before being used in a fermentation industry in order to ascertain how much of the various nutrients must be added. A raw material with extremes of variability in quality is undesirable as extra costs are needed. - Analysis of the raw material, - Nutrients which may need to be added to attain the usual and expected quality in the medium.

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Adequate chemical composition of medium The medium must have adequate amounts of C, N, minerals and vitamins in the appropriate quantities and proportions necessary for the optimum production of the commodity in question. T he compounds in the medium must utilizable by the organisms. Thus most yeasts utilize hexose sugars, whereas only a few will utilize lactose. Cellulose is not easily used and is utilized only by a limited number of organisms. Some organisms grow better in one or the other substrate. Fungi will for instance readily grow in corn steep liquor while actinomycetes will grow more readily on soya bean cake.

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Presence of relevant precursors Precursors necessary for the synthesis of the finished product. Precursors often stimulate production of secondary metabolites either by - increasing the amount of a limiting metabolite, - by inducing a biosynthetic enzyme or both. Precursors include amino acids & small molecules. For penicillin G to be produced the medium must contain a phenyl compound. Corn steep liquor contains phenyl precursors. Other precursors are cobalt in media for Vitamin B12 production & chlorine for the chlorine containing antibiotics, chlortetracycline, & griseofulvin (Fig. 4.1).

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Satisfaction of growth and production requirements of the microorganisms Many industrial organisms have two phases of growth in batch cultivation: the phase of growth, or the trophophase, and the phase of production, or the idiophase. In the first phase cell multiplication takes place rapidly, with little or no production of the desired material. It is in the second phase that production of the material takes place, usually with no cell multiplication and following the elaboration of new enzymes. Often these two phases require different nutrients or different proportions of the same nutrients.

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The medium must be complete and be able to cater for these requirements. For example high levels of glucose and phosphate inhibit the onset of the idiophase in the production of a number of secondary metabolites of industrial importance. The levels of the components added must be such that they do not adversely affect production.

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Corn steep liquor This is a by-product of starch manufacture from maize. As a nutrient for most industrial organisms corn steep liquor is considered adequate, rich in carbohydrates, nitrogen, vitamins, and minerals. highly acidic, it must be neutralized (usually with CaCO 3 ) before use.

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Pharmamedia Yellow fine powder made from cotton-seed embryo. It is used in the manufacture of tetracycline and some semi-synthetic penicillins. rich in protein, (56% w/v) and contains 24% carbohydrate, 5% oil, and 4% ash rich in calcium, iron, chloride, phosphorous, and sulfate.

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Distillers soluble By-product of the distillation of alcohol from fermented grain. (maize or barley) It is rich in nitrogen, minerals, and growth factors.

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Soya bean meal The seeds are heated before being extracted for oil that is used for food, as an antifoam in industrial fermentations, or used for the manufacture of margarine. The resulting dried material, soya bean meal, has about 11% nitrogen, and 30% carbohydrate and may be used as animal feed. Its nitrogen is more complex than that found in corn steep liquor Not readily available to most microorganisms, except Actinomycetes. It is used particularly in tetracycline and streptomycin fermentations.

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It is a by-product of the sugar industry. For the production of cells the variability in molasses quality is not critical. For metabolites such as citric acid, it is very important as minor components of the molasses may affect the production of these metabolites. High test’ molasses (inverted molasses) is a brown thick syrup liquid used in the distilling industry and containing about 75% total sugars (sucrose and reducing sugars) and about 18% moisture.

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Indeed it is invert sugar, (i.e reducing sugars resulting from sucrose hydrolysis). Produced by hydrolysis of the concentrated juice with acid. In the so-called Cuban method, invertase is used for the hydrolysis. Sometimes ‘A’ sugar may be inverted and mixed with ‘A’ molasses.

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Sulfite Liquor Sulfite liquor (also called waste sulfite liquor,) is the aqueous effluent resulting from the sulfite process for manufacturing cellulose or pulp from wood. During the sulfite process, hemicelluloses hydrolyze and dissolve to yield the hexose sugars, glucose, mannose, galactose, fructose and the pentose sugars, xylose, and arabinsoe. Used as a medium for the growth of microorganisms after being suitably neutralized with CaCO3 and enriched with ammonium salts or urea, and other nutrients. It has been used for the manufacture of yeasts and alcohol. Some samples do not contain enough assaimilable carbonaceous materials for some modern fermentations. They are therefore often enriched with malt extract, yeast autolysate, etc.

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GROWTH FACTORS Not synthesized by the organism Must be added to the medium. Function as cofactors of enzymes and may be vitamins, nucleotides etc. The pure forms are usually too expensive for use in industrial media Growth factors are required only in small amounts.

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WATER Water is a raw material of vital importance in industrial microbiology. Major component of the fermentation medium. Cooling, washing and cleaning. It is used in large quantities. In some industries the quality of the product depends to some extent on the water. To ensure constancy of product quality the water must be regularly analyzed for minerals, color, pH, etc. and adjusted as may be necessary. Due to the importance of water, in situations where municipal water supplies are likely to be unreliable, industries set up their own supplies.

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SOME POTENTIAL SOURCES OF COMPONENTS OF INDUSTRIAL MEDIA The materials to be discussed are mostly found in the tropical countries. Any microbiological industries to be sited must use the locally available substrates. Carbohydrate Sources Polysaccharides that have to be hydrolyzed to sugar before being used. (a) Cassava (manioc) The roots of the cassava-plant Manihot esculenta Crantz (food & feed) in the tropical world. High yielding, little attention when cultivated, and the roots can keep in the ground for many months without deterioration before harvest. The inner fleshy portion is a rich source of starch and has served, after hydrolysis, as a carbon source for single cell protein, ethanol. In Brazil it is one of the sources of ethanol production.

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(b) Sweet potato Ipomca batatas is a warm-climate crop. It can be grown also in sub tropical regions. Large number of cultivars vary in the colors of the tuber flesh and of the skin; they also differ in the tuber size, time of maturity, yield, and sweetness. They are widely grown in the world. Regarded as minor sources of carbohydrates in comparison to wheat, or cassava. Do not require much agronomic attention.

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Used as sources of sugar on a semi-commercial basis. The fleshy roots contain saccharolytic enzymes. The syrup made from boiling the tubers has been used as a carbohydrate (sugar) source in compounding industrial media. Butyl alcohol, acetone and ethanol have been produced from such a syrup, and in quantities higher than the amounts produced from maize syrup of the same concentration. Not widely consumed as food, it is possible that it may be profitable to grow them for industrial microbiology media as well as for the starch industry. Some variety can yields up to 40 tonnes per hectare, a much higher yield than cassava or maize.

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(c) Yams Yams (Dioscorea spp) are widely consumed in the tropics. Compared to other tropical Cultivation is tedious. Enough of this tuber is not produced even for human food. It is inconceivable to suggest for growing solely for use in compounding industrial media. Yams have been employed in producing various products such as yam flour and yam flakes. Mass production may encourage its use or its wasted peelings as industrial microbiological media.

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(d) Cocoyam Cocoyam is a blanket name for several edible members of the monocotyledonous (single seed-leaf) plant of the family Araceae (the aroids), the best known two genera of which are Colocasia (tano) and Xanthosoma (tannia). They are grown and eaten all over the tropical world. Laborious to cultivate, require large quantities of moisture and do not store well. They are not the main source of carbohydrates in regions where they are grown. Cocoyam starch has been found to be of acceptable quality for pharmaceutical purposes. Should it find use in that area, starchy by-products could be hydrolyzed to provide components of industrial microbiological media.

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(e) Millets This is a collective name for several cereals whose seeds are small in comparison with those of maize, sorghum, rice, etc. The plants are also generally smaller. They are classified as the minor cereals as they generally do not form major components of human food. They are hardy and will tolerate great drought and heat, grow on poor soil and mature quickly. It could become potential sources of cereal for use in industrial microbiology media. Millets are grown all over the world in the tropical and sub- tropical regions and belong to various genera: Pennisetum americanum (pearl or bulrush millet), Setaria italica (foxtail millet), Panicum miliaceum (yard millet), Echinochloa frumentacea (Japanese yard millet) and Eleusine corcana (finger millet).

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Millet starch has been hydrolyzed by malting for alcohol production on an experimental basis as far back as 50 years ago. (f) Rice Rice, Oryza sativa is one of the leading food corps of the world, especially in the tropical areas. High-cost commodity. Ease of mechanization, storability. Availability of improved seeds. The increase in rice production is expected to become so efficient for industrial microbiological use. Rice is used as brewing adjuncts and has been malted experimentally for beer brewing.

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(g) Sorghum Sorghum, Sorghum bicolor, is the fourth in term of quantity of production of the world’s cereals, after wheat, rice, and corn. It is used for the production of special beers in various parts of the world. It has been mechanized and has one of the greatest potential among cereals for use as a source of carbohydrate in industrial media in regions of the world where it thrives. It has been successfully malted and used in an all- sorghum lager beer which compared favorably with barley lager beer

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(h) Jerusalem artichoke Jerusalem artichoke, Helianthus tuberosus, is a member of the plant family compositae, where the storage carbohydrate is inulin, a polymer of fructose into which it can be hydrolyzed. It is a root-crop and grows in temperate, semi- tropical and tropical regions.

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Protein Sources (a) Peanut (groundnut) meal Various leguminous seeds. Only peanuts (groundnuts) Arachis hypogea will be discussed. The nuts are rich in liquids and proteins. The groundnut cake left after the nuts have been freed of oil is often used as animal feed. Oil from peanuts may be used as anti-foam while the press-cake could be used for a source of protein.

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(b) Blood meal Blood consists of about 82% water, 0.1% carbohydrate, 0.6% fat, 16.4% nitrogen, and 0.7% ash. It is a waste product in abattoirs although it is sometimes used as animal feed. Drying is achieved by passing live steam through the blood until the temperature reaches about 100°C. This treatment sterilizes it and also causes it to clot. It is then drained, pressed to remove serum, further dried and ground. The resulting blood-meal is chocolate-colored and contains about 80% protein and small amounts of ash and lipids.

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(c) Fish Meal Fish meal is used for feeding farm animals. It is rich in protein (about 65%) and, minerals (about 21% calcium 8%, and phosphorous 3.5%) and may therefore be used for industrial microbiological media production. Fish meal is made by drying fish with steam either aided by vacuum or by simple drying. Alternatively hot air may be passed over the fish placed in revolving drums. It is then ground into a fine powder.

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THE USE OF PLANT WASTE MATERIALS IN INDUSTRIAL MICROBIOLOGY MEDIA: SACCHARIFICATION OF POLYSACCHARIDES Agriculture waste materials and even crops. Plentiful and renewable. Large amounts of polysaccharides which are in need for hydrolysis or saccharification to be utilizable by industrial microorganisms. Hydrolyzed polysaccharides may give more available sugars for microorganisms. The sugars could be converted into ethanol for example or any other commodity produced by Mos.

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Starch It is a mixture of two polymers of glucose: amylose and amylopectin. Amylose is a linear (1-4) – D glucan usually having a degree of polymerization (D.P., i.e. number of glucose molecules) of about 400 and having a few branched residues linked with (1-6) linkages. Amylopectin is a branched D glucan with predominantly – D (1-4) linkages and with about 4% of the – D (1-6) type (Fig. 4.3). Amylopectin consists of amylose – like chains of D. P. 12 – 50.

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Starches differ in their proportion of amylopectin and amylose according to the source. The common type of maize, for example, has about 26% of amylose and 74% of amylopectin. Others may have 100% amylopectin and still others may have 80 – 85% of amylose.

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Saccharification of starch Starch occurs in discrete crystalline granules in plants, and in this form is highly resistant to enzyme action. However when heated to about 55°C – 82°C depending on the type, starch gelatinizes and dissolves in water and becomes subject to attack by various enzymes. Before saccharification, the starch or ground cereal is mixed with water and heated to gelatinize the starch and expose it to attack by the saccharifying agents. The gelatinization temperatures of starch from various cereals is given in Table 12.1. The saccharifying agents used are dilute acids and enzymes from malt or microorganisms.

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Saccharification of starch with acid The starch-containing material to be hydrolyzed is ground and mixed with dilute hydrochloric acid, sulfuric acid or even sulfurous acid. When sulfurous acid is used itcan be introduced merely by pumping sulfur dioxide into the mash.

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The concentrations of the mash and the acid, length of time and temperature of the heating have to be worked out for each starch source. The actual composition of the hydrolysate will depend on the factors mentioned above. Starch concentration is particularly important: if it is too high, side reactions may occur leading to a reduction in the yield of sugar. At the end of the reaction the acid is neutralized.

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Use of enzymes Collectively diastase. They are now called amylases. Advantages over acids: (a) since the pH for enzyme hydrolysis is about neutral, there is no need for special vessels which must stand the high temperature, pressure, and corrosion of acid hydrolysis; (b) enzymes are more specific and hence there are fewer side reactions leading therefore to higher yields; (c) acid hydrolysis often yields salts which may have to be removed constantly or periodically thereby increasing cost; (d) it is possible to use higher concentrations of the substrates with enzymes than with acids.

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Enzymes involved in the hydrolysis of starch They are divisible into six groups. (i) Enzymes that hydrolyse α – 1, 4 bonds and by-pass α – I, 6 bonding: The typical example is α - amylase. This enzyme hydrolyses randomly the inner α - (1 4) - D - glucosidic bonds of amylose and amylopectin (Fig. 4.3). The cleavage can occur anywhere as long as there are at least six glucose residues on one side and at least three on the other side of the bond to be broken. The result is a mixture of branched - limit dextrins (i.e., fragments resistant to hydrolysis and contain the α - D (1-6) linkage (Fig. 4.4) derived from amylopectin) and linear glucose residues especially maltohexoses, maltoheptoses and maltotrioses. α -Amylases are found in virtually every living cell and the property and substrate pattern of α - amylases vary according to their source. Animall α - amylases in saliva and pancreatic juice completely hydrolyze starch to maltose and D-glucose. Among microbial α - amylases some can withstand temperatures near 100°C.

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(ii) Enzymes that hydrolyse the α–1, 4 bonding, but cannot by-pass the α– 1,6 bonds: Beta amylase: This was originally found only in plants but has now been isolated from micro-organisms. Beta amylase hydrolyses alternate α– 1,4 bonds sequentially from the non-reducing end (i.e., the end without a hydroxyl group at the C – 1 position) to yield maltose (Figs. 4.3 and 4.5). Beta amylase has different actions on amylose and amylopectin, because it cannot by-pass the α –1:6 – branch points in amylopectin. Therefore, while amylose is completely hydrolyzed to maltose, amylopectin is only hydrolyzed to within two or three glucose units of the α– 1.6 - branch point to yield maltose and a ‘beta-limit’ dextrin which is the parent amylopectin with the ends trimmed off.

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Debranching enzymes (see below) are able to open up the α– 1:6 bonds and thus convert beta-limit dextrins to yield a mixture of linear chains of varying lengths; beta amylase then hydrolyzes these linear chains. Those chains with an odd number of glucose molecules are hydrolyzed to maltose, and one glucose unit per chain. The even numbered residues are completely hydrolyzed to maltose. In practice there is a very large population of chains and hence one glucose residue is produced for every two chains present in the original starch.

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(iii) Enzymes that hydrolyze (α —1, 4 and α — 1:6 bonds: The typical example of these enzymes is amyloglucosidase or glucoamylase. This enzyme hydrolyzes α - D - (1-4) -D – glucosidic bonds from the non-reducing ends to yield D – glucose molecules. When the sequential removal of glucose reaches the point of branching in amylopectin, the hydrolysis continues on the (1-6) bonding but more slowly than on the (1-4) bonding. Maltose is attacked only very slowly. The end product is glucose. (iv) De-branching enzymes: At least two de-branching enzymes are known: pullulanase and iso-amylase.

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Pullulanase: This is a de-branching enzyme which causes the hydrolysis of α — D – (1 6) linkages in amylopectin or in amylopectin previsouly attacked by alphaamylase. It does not attack α - D (1-4) bonds. However, there must be at least two glucose units in the group attached to the rest of the molecules through an α -D- (1-6) bonding. Iso-amylase: This is also a de-branching enzyme but differs from pullulanase in that three glucose units in the group must be attached to the rest of the molecules through an α - D – (1 6) bonding for it to function. (v) Enzymes that preferentially attack α - 1, 4 linkages: Examples of this group are glucosidases. The maltodextrins and maltose produced by other enzymes are cleaved to glucose by - glucosidases.

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They may however sometime attack unaltered polysaccharides but only very slowly. (vi) Enzymes which hydrolyze starch to non-reducing cyclic D-glucose polymers known as cyclodextrins or Schardinger dextrins: Cyclic sugar residues are produced by Bacillus macerans. They are not acted upon by most amylases although enzymes in Takadiastase produced by Aspergillus oryzae can degrade the residues. Industrial saccharification of starch by enzymes In industry the extent of the conversion of starch to sugar is measured in terms of dextrose equivalent (D.E.). This is a measure of the reducing sugar content, determined under defined conditions involving Fehling’s solution.

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The D.E is calculated as percentage of the total solids. Acid is being replaced more and more by enzymes. Sometimes acid is used initially and enzymes employed later. Practical upper limit of acid saccharification is 55 D.E. Beyond this, breakdown products begin to accumulate. Furthermore, with acid hydrolysis reversion reactions occur among the sugar produced. These two withdraws are avoided when enzymes are utilized. By selecting enzymes specific sugars can be produced.

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Industrialy used enzymes are produced in germinated seeds and by micro-organisms. Barley malt is widely used for the saccharification of starch. It contains large amounts of various enzymes notably -amylase and - glucosidase which further split saccharides to glucose. All the enzymes discussed above are produced by different micro-organisms and many of these enzymes are available commercially. The most commonly encountered organisms producing these enzymes are Bacillus spp, Streptomyeces spp, Aspergillus spp, Penicillium spp, Mucor spp and Rhizopus spp.

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Cellulose, Hemi-celluloses and Lignin in Plant Materials Cellulose Cellulose is the most abundant organic matter on earth. Does not exist pure in nature and even the purest natural form (that found in cotton fibres) contains about 6% of other materials. Three major components, cellulose, hemi-cellulose and lignin occur roughly in the ratio of 4:3:3 in wood.

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Hemicelluloses Group of carbohydrates whose main and common characteristic is that they are soluble in, and hence can be extracted with, dilute alkali. They can then be precipitated with acid and ethanol. They are very easily hydrolyzed by chemically or biologically. The nature of the hemicellulose varies among plants. In cotton the hemicelluloses are pectic substances, which are polymers of galactose. In wood, they consist of short (DP less than 200) branched heteropolymers of glucose, xylose, galactose, mannose and arabinose as well as uronic acids of glucose and galactose linked by 1 – 3, 1 – 6 and 1 – 4 glycosidic bonding.

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Lignin Lignin is a complex three-dimensional polymer formed from cyclic alcohols. (Fig. 4.6). It is important because it protects cellulose from hydrolysis. Cellulose is found in plant cell-walls which are held together by a porous material known as middle lamella. In wood the middle lamella is heavily impregnated with lignin which is highly resistant and thus protects the cell from attack by enzymes or acid.

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Pretreatment of cellulose-containing materials before saccharification In order to expose lignocellulosics to attack, a number of physical and chemical methods are in use, or are being studied, for altering the fine structure of cellulose and/or breaking the lignin- carbohydrate complex. Chemical methods include the use of swelling agents such a NaOH, some amines, concentrated H 2 SO 4 or HCI or proprietary cellulose solvents such as ‘cadoxen’ (tris thylene-diamine cadmium hydroxide).

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These agents introduce water between or within the cellulose crystals making subsequent hydrolysis, easier. Steam has also been used as a swelling agent. The lignin may be removed by treatment with dilute H 2 SO 4 at high temperature. Physical methods of pretreatment include grinding, irradiation and simply heating the wood. Hydrolysis of cellulose After pretreatment, wood may be hydrolyzed with dilute HCI, H 2 SO 4 or sulfites of Ca, Mg or Na under high temperature and pressure. When, however, the aim is to hydrolyze wood to sugars, the treatment is continued for longer than is done for paper manufacture.

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Enzymatic hydrolysis has been subjected to many research and work. Fungi was the main source of cellulolytic enzymes. Trichoderma viride and T. koningii have been the most efficient cellulase producers. Penicillicum funiculosum and Fusarium solani have also been shown to possess potent cellulases. Cellulase has been resolved into at least three components: C1, Cx, and -glucosidases. The C1 component attacks crystalline cellulose and loosens the cellulose chain, after which the other enzymes can attack cellulose.

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Cx enzymes are β - (1 4) glucanases and hydrolyse soluble derivatives of cellulose or swoollen or partially degraded cellulose. Their attack on the cellulose molecule is random and cellobiose (2-sugar) and cellotroise (3-sugar) are the major products. Enzymes may also act by removing successive glucose units from the end of a cellulose molecule. β-glucosidases hydrolyze cellobiose and short-chain oligo- saccharides derived from cellulose to glucose, but do not attack cellulose. They are able to attack cellobiose and cellotriose rapidly. Many organisms described in the literature as ‘cellulolytic’ produce only Cx and -glucosidases because they were isolated initially using partially degraded cellulose. The four organisms mentioned above produce all three members of the complex

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Molecular structure of cellulose Cellulose is a linear polymer of D-glucose linked in the Beta-1, 4 glucosidic bondage. The bonding is theoretically as vulnerable to hydrolysis as the one in starch. However, cellulose – containing materials such as wood are difficult to hydrolyze because of: (a) the secondary and tertiary arrangement of cellulose molecules which confers a high crystallinity on them and (b) the presence of lignin. The degree of polymerization (D. P.) of cellulose molecule is variable, but ranges from about 500 in wood pulp to about 10,000 in native cellulose.

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When cellulose is hydrolyzed with acid, a portion known as the amorphous portion which makes up 15% is easily and quickly hydrolyzed leaving a highly crystalline residue (85%) whose DP is constant at 100-200. The crystalline portion occurs as small rod-like particles which can be hydrolyzed only with strong acid. (Fig. 4.7)